Chromatography is an analytical technique for separating a mixture into its constituents. The mixture containing compounds is moved through a stationary phase. The compounds held in the mixture, the mobile phase, exhibit different affinity for the stationary phase. The compounds are thus retained or allowed to pass at different rates as the mobile phase moves producing bands or peaks of the compounds in the stationary phase or exit the stationary phase at different times.
Gas chromatography uses one or more mixtures comprising a gas as a solvent to carry sample compounds to be analyzed. Liquid chromatography uses one or more mixtures of liquids as a solvent. Supercritical fluid chromatography uses one or more gases held at pressures and temperature in which the gas takes on one or features of liquids as a solvent. This document will use the term “fluid” to means gases, liquids and supercritical fluids.
A common form of liquid chromatography, high performance liquid chromatography (HPLC), uses pressure to propel liquids through a stationary phase. In a typical HPLC analysis, a solvent (mobile phase) is pumped through a column packed with a solid porous material (stationary phase). A sample is injected into the solvent and compounds in the sample are adsorbed on the stationary phase. This step of placing the sample in the stationary phase is referred herein as “loading”. Each compound exhibits different affinity to the stationary phase so that they exit the column separated in time. The step or process of leaving the stationary phase is referred herein as “elution” and the materials leaving the stationary phase are referred to as the “eluant.” A detector receives the column eluent and generates an electrical signal indicative of the presence of compounds in the sample. A mass spectrometer is frequently used as a detector because it may provide detailed information about the chemical identity of the compounds.
Chromatographers desire an efficient separation with respect to the time taken for a separation, materials consumed and the degree of separation. Efficiency may be increased and the time reduced by gradient elution, in which a mixture of two or more solvents is used as the mobile phase. The proportions of the two solvents is varied as the separation proceeds, typically to change the polarity to change the interaction of the compounds with the stationary phase. A change of the proportions of the two solvents may cause one or more compounds to elute and thereby shorten the retention time for such compounds.
Method of generating solvent gradients with a variety of pump means comprising multiple pumps, single pumps with proportioning valves are known in the art. See: Berry, Schwartz, J. Chrom. Sci. 1989 vol. 45 pp 67-116, the contents of which are incorporated herein by reference.
The quest for better sensitivity has led to the use of even smaller diameter columns employing even smaller particle sizes. Such columns require pumps capable of producing very low stable flows at very high pressures. For example, current column technology (known as nanoflow) may use a 75 μm diameter capillary column, and particles of 1.7 μm diameter, requiring a flow often less than 500 nl/minute at a pressure as high as 8000 psi.
When gradient elution is performed at low flow rates, the delay between the initiation of the gradient by the pumps and the time at which it appears at the column inlet, referred herein as “gradient delay”, becomes a major cause of extended analysis times. For example, if the dead volume of the connections between the pumps, sample injection valve and the column inlet is 5 μl, at a flow rate of 250 nl/minute, there is a gradient delay of twenty minutes. During the twenty minutes of delay, sample is not presented to the detector and the detector is not generating data. Some detectors are very expensive, such as mass spectrometers.
It is desirable to maximize the amount of sample presented to such detectors to generate data as efficiently as possible.